Li et al. Molecular Cytogenetics (2016) 9:6 DOI 10.1186/s13039-016-0217-0

RESEARCH Open Access Molecular cytogenetic characterization of breviaristatum chromosomes in background revealing the genomic divergence between Dasypyrum Guangrong Li, Dan Gao, Hongjun Zhang, Jianbo Li, Hongjin Wang, Shixiao La, jiwei Ma and Zujun Yang*

Abstract Background: The uncultivated species Dasypyrum breviaristatum carries novel diseases resistance and agronomically important genes of potential use for wheat improvement. The development of new wheat-D. breviaristatum derivatives lines with disease resistance provides an opportunity for the identification and localization of resistance genes on specific Dasypyrum chromosomes. The comparison of wheat-D. breviaristatum derivatives to the wheat-D. villosum derivatives enables to reveal the genomic divergence between D. breviaristatum and D. villosum. Results: Themitoticmetaphaseofthewheat-D. breviaristatum partial amphiploid TDH-2 and durum wheat -D. villosum amphiploid TDV-1 were studied using multicolor fluorescent in situ hybridization (FISH). We found that the distribution of FISH signals of telomeric, subtelomeric and centromeric regions on the D. breviaristatum chromosomes was different from those of D. villosum chromosomes by the probes of Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7 and Oligo-pHv62-1. A wheat line D2139, selected from a cross between wheat lines MY11 and TDH-2, was characterized by FISH and PCR-based molecular markers. FISH analysis demonstrated that D2139 contained 44 chromosomes including a pair of D. breviaristatum chromosomes which had originated from the partial amphiploid TDH-2. Molecular markers confirmed that the introduced D. breviaristatum chromosomes belonged to homoeologous group 7, indicating that D2139 was a 7Vb disomic addition line. The D2139 displayed high resistance to wheat stripe rust races at adult stage , which may be inherited from, D. breviaristatum chromosome 7Vb. Conclusion: The study present here revealed that the large divergence between D. breviaristatum and D. villosum with respected to the organization of different repetitive sequences. The identified wheat- D. breviaristatum chromosome addition line D2139 will be used to produce agronomically desirable germplasm for wheat breeding. Keywords: Dasypyrum breviaristatum, Fluorescence in situ hybridization, Molecular marker, Wheat

Background VVVbVb. Dasypyrum species possess agronomically im- The Dasypyrum (or Haynaldia) consists of only portant genes such as disease resistance, high protein two diploid species, the annual and quality and drought tolerance, all of which represent valu- perennial D. breviaristatum [1]. The genomes of diploid able resources for global wheat breeding [3]. The species D. villosum and D. breviaristatum were assigned the sym- D. villosum has been extensively hybridized with wheat for bols V and Vb, respectively [2, 3]. Based on the sequences at least 6 decades, and several disease resistance genes comparison of nr5S DNA multigene family, Baum et al. have been successfully transferred to wheat [5–7]. Above [4] suggested that the genome constitution of 4x D. all, over 20 elite cultivars carrying the wheat- D. villosum breviaristatum should be considered as an allotetraploid chromosome T6AL · 6VS translocation with powdery mildew resistance gene Pm21 have been released into agri- cultural production in China [8, 9]. Given the widespread * Correspondence: [email protected] School of Life Science and Technology, University of Electronic Science and success of this introgression from D. villosum, researches Technology of China, Chengdu 610054, China

© 2016 Li et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Molecular Cytogenetics (2016) 9:6 Page 2 of 9

have been conducted with a similar aim to transfer useful be related to the presence of D. breviaristatum chro- genes from D. breviaristatum into wheat. Subsequently, mosomes. Each of the seven pairs of D. breviaristatum the wheat- D. breviaristatum partial amphiploid [10] and chromosomes were also distinguished using probes Oligo- wheat- D. breviaristatum introgression lines with multiply pSc119.2, Oligo-pTa535 in TDH-2 (Fig. 1b). These chro- disease resistances have developed [11, 12]. mosomes were temporarily named Vb1-Vb7 (Fig. 1b). Molecular and cytogenetic methods have been previ- FISH using probes Oligo-pSc119.2, Oligo-pTa535, Oligo- ously employed to assess the level of chromosomal di- (GAA)7 were also carried out on the chromosomes of the vergence of these two Dasypyrum species [13, 14]. A Triticum turgidum cv. Jorc-69- D. villosum amphiploid large number of interspecific and intraspecific chromo- TDV-1(Fig.1candd).Wefoundthatthesignalsofprobe some variations and significant genomic diversification Oligo-pSc119.2 were mainly located on terminal sites, were observed among different Dasypyrum accessions, while the hybridization signals of Oligo-pTa535 were probably due to the out-crossing characteristic of these distributed along the chromosome arms of D. villosum. two Dasypyrum species [2]. Each pair of Dasypyrum The probe Oligo- (GAA)7 hybridized to 2V-7V of D. villo- chromosomes were transferred into a wheat background sum chromosomes at their centromeric or sub-terminal after intergeneric hybridizations. The wheat- Dasypyrum regions. Moreover, we produced a high tandem repeat chromosome addition lines with different Dasypyrum sequences probe Oligo-pHv62-1 as reported by Li et al. species or accession origins allow comparison of the [17]. FISH revealed that Oligo-pHv62-1 present in ter- different Dasypyrum genomes in wheat backgrounds. In minal or sub-terminal heterochromatic C-banding regions the present study, fluorescent in situ hybridization (FISH) of D. villosum chromosomes in TDV-1, but was absent in was carried out to characterize differences between D. bre- D. breviaristatum chromosomes of TDH-2 (Fig. 1e and f). viaristatum and D. villosum chromosomes by comparing The comparative FISH karyotypes of the D. breviarista- karyotypes between the wheat- D. breviaristatum partial tum and D. villosum chromosomes allows easily to distin- amphiploid TDH-2 and Triticum turgidum - D. villosum guish each individual Dasypyrum chromosome in wheat amphiploid TDV-1, The FISH and molecular markers background. were applied to identify new wheat- D. breviaristatum addition line with stripe rust resistance, which will be a FISH of wheat- D. breviaristatum addition line D2139 useful germplasm for wheat genetics and breeding. Sequential multi-color ND-FISH by probes Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7 was conducted to charac- Results terize the mitotic metaphase cells of D2139 (Fig. 2). Comparative FISH karyotype of TDH-2 and TDV-1 The chromosome number of D2139 is 2n = 44, including The mitotic metaphase chromosomes of the wheat- D. all the 42 wheat chromosomes and two alien chromo- breviaristatum partial amphiploid TDH-2, were hybridized somes added in the wheat background. The probes Oligo- with probes Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7 pSc119.2, Oligo-pTa535, showed a pair of chromosomes by sequential multicolor-FISH (Fig. 1). As shown in with faint Oligo-pSc119.2 hybridization signals at the telo- Fig. 1a, the FISH hybridization signals of the probes meric region of long arm, and strong hybridization signals Oligo-pSc119.2 and Oligo-pTa535 can easily identify of Oligo-pTa535 along the long and short arm in D2139 the 28 wheat chromosomes from 1A-7A and 1B-7B (Fig. 2). The FISH hybridization pattern of the chromo- based on the standard FISH karyotype of wheat chromo- somes was identical to D. breviaristatum chromosomes somes using the same probes described by Tang et al. [15]. Vb7 (Fig. 1). Therefore, we concluded that the line D2139 Yang et al. [10] reported that the partial amphiploid TDH-2 was a chromosome Vb7 addition line. Comparing the was produced by the elimination of some chromosomes FISH patterns of D2139 parents MY11 [15] and TDH-2 fromthewheatChineseSpring(CS)-D. breviaristatum (Fig. 1), it appeared that the D2139 line inherited the A decaploid amphiploid. It is likely that the A and B chro- and B- genome chromosomes from MY11 and/or TDH-2. mosomes of TDH-2 originated from CS. By comparing Based on the FISH patterns, D2139 inherited chromosomes the FISH patterns of Oligo-pSc119.2 and Oligo-pTa535 5A, 7A, 1B, and 7B which were identical to the TDH-2 par- probes of TDH-2 to those of CS by Tang et al. [15], we ent, while 6B, 2B appeared to be from MY11. Since there is found additional signals corresponding to probe Oligo- no D-genome in the partial amphiploid TDH-2, D2139 pSc119.2 on the terminal regions of 1BS and 2BL in would have inherited D-chromosomes from MY11. As TDH-2 (Fig. 1b). After comparing the (GAA)n signal shown in Fig. 2c, chromosomes 1D and 3D revealed clear distribution on TDH-2 chromosomes with those of CS differences in the distribution of Oligo-pSc119.2 signals reported by Danilova et al. [16], we observed that the compared to previously published FISH patterns of D- (GAA)n signals on chromosome 7A of CS were absent in genome chromosomes of MY11 [15]. The terminal region TDH-2. The results suggest that at least three wheat chro- of 1DL showed strong Oligo-pSc119.2 signals, while the mosomes have undergone structural change(s) which may Oligo-pSc119.2 signals were absent from the 3DS terminal Li et al. Molecular Cytogenetics (2016) 9:6 Page 3 of 9

Fig. 1 FISH and karyotypes of TDH-2 (a, b, f)andTDV-1(c, d, e). The mitotic metaphase chromosomes (a, c) after hybridization with probes Oligo-pTa535

(red) and Oligo-pSc119.2 (green), or (b, d) hybridized with Oligo-(GAA)7 (red) The boxes shows the modified chromosomes compared to its parents. Figures e and f are hybridized by probe of pHv62-3 (green) region. The observation implies that the transmission of that they would presumably amplify fragments from the the D. breviaristatum chromosomes may be associated with corresponding linkage group(s) of wheat genomes [18]. structural changes in wheat chromosomes. Our previous studies showed that the PLUG markers were useful for producing alien chromosome-specific markers Molecular markers analysis [19, 20]. A total of 21 PLUG markers from wheat homo- PLUG primers were designed from rice genomic DNA se- eologous group 7 were tested on D2139 compared to quences specific for the syntenic regions, in the expectation its parents MY11 and TDH-2 (Table 1). Based on the Li et al. Molecular Cytogenetics (2016) 9:6 Page 4 of 9

Fig. 2 Sequential FISH karyotypes of D2139. Left figure (a) was stained with DAPI (blue), Oligo-pTa535 (red) and Oligo-pSc119.2 (green), while the right figure (b) chromosomes were stained by PI (red) and Oligo-GAA (green). The box shows the modified chromosomes compared to the parent line MY11 (c) amplification of nulli-tetrasomic lines of Chinese Spring, Fig. 4a, the spike length of D2139 was 11–12 cm, which the PLUG markers give rise to the 7A, 7B and 7D specific was longer than the wheat parents (9–10 cm), while the bands, respectively (Fig. 3). A total of 13 pairs of primers D2139 had 22–22 spikelets per spike, closely resembling generated the identical bands from D. breviaristatum VbVb MY11. When inoculated with P. striiformis f. sp. tritici and TDH-2 to those from the disomic addition line D2139. (PST) races CYR32 and CYR33 at adult plant stage, the These results suggested that the D. breviaristatum chromo- TDH-2 and D2139 lines were highly resistance to the iso- some in D2139 belonged to homoeologous group 7. Five lates, whereas wheat MY11 and CS were highly susceptible markers out of the 13 had previously been mapped onto (Fig. 4b). These results indicated that the stripe rust resist- the short arms and eight mapped onto the long arms of ance in D2139 was from the TDH-2 parent, and originates group 7 (Table 1). After combining these PCR results with from D. breviaristatum. the FISH patterns, we concluded that the D2139 was a 7Vb addition line, and the Vb7 was chromosome 7Vb.The Discussion wheat CS - D. villosum 7V addition line TA7683 was also With respect to the genomic relationship between two used to test the PLUG markers. We found that seven of 13 Dasypyrum species, D. breviaristatum and D. villosum, primer pairs showed polymorphic amplification differences cytogenetic and molecular evidence has revealed the huge between the D. breviaristatum 7Vb and D. villosum 7V genetic divergence between the two species. Friebe et al. chromosomes (Table 1). The results suggested that se- [21] established the C-banded patterns of D. villosum,and quence divergence may have occurred among Dasy- then Linde-Laursen and Frederiksen [22] observed exten- pyrum species during their evolution. sive C-band karyotype differences between the two genomes. De Pace et al. [23] isolated a repeat sequence Agronomic traits and rust resistance analysis that was mapped further distally on D. villosum chromo- The spike phenotype and stripe rust resistance of lines somes using FISH compared to wheat chromosomes. D2139, TDH-2, CS and MY11 were observed. As shown in Galasso et al. [13] found that the differentiated GISH Li et al. Molecular Cytogenetics (2016) 9:6 Page 5 of 9

Table 1 The primers used to localize the Vb7 specific amplification in D2139 No. Primers name Primer sequences (5’–3’) Wheat bin map Restriction enzyme Length of Vb7 bands 1 TNAC1776 F: ATCATCCTGCTGCTACTGTGC 7AS2-0.73–0.83 – R: CCTTCTCAGCTTAGCGATGTG 4AL2-0.75–0.80 7DS4-0.73–1.00 2 TNAC1782 F: TCACTGAACAGCCTAGACATGG 7AS2-0.73–0.83 HaeIII 690 bp R: ATTCGCAGACCGCATCTATC 7BS2-0.27–1.00 7DS4-0.73–1.00 3 TNAC1803 F: TGCGACCAGTCTCTTTGAAAT C-7AL1-0.39 HaeIII 800 bp* R: GTCGGAGCCTGGATCTCTAGT 7BL2-0.38–0.63 7DL5-0.30–0.61 4 TNAC1806 F: ATTCCTCGTGAATTGCTGGAT 7AS8-0.45–0.59 TaqI 350 bp* R: TCTGCAGTTAGGGACTTGAAA 7BS2-0.27–1.00 7DS2-0.61–0.73 5 TNAC1811 F: CTGCTCAACGAGTTCATCGAC 7AL1-0.39–0.63 TaqI 740 bp R: TTGGAGTGGACGTTGCATT 7BL2-0.38–0.63 7DL5-0.30–0.61 6 TNAC1812 F: ACTTCGCTTGGTCTCCTCAAT 7AL5-0.63–0.71 TaqI 860 bp R: GAGAAGTGTGCCAATTCCAAA 7BL7-0.63–0.78 7DL5-0.30–0.61 7 TNAC1815 F: AGCAGACATCAGCAAGTTTGAG 7AL1-0.39–0.63 TaqI 600 bp* R: ACTGACAAGCCCATGATTGAC 7BL2-0.38–0.63 7DL5-0.30–0.61 8 TNAC1822 F: CCCTCCGTCCGTGCAAAT 7AL5-0.63–0.71 TaqI 730 bp* R: GGCTGATGATGGAGACGTG 7BL2-0.38–0.63 7DL2-0.61–0.82 9 TNAC1867 F: GCCTTTCCTTTGGTAGTCTGG C-7AL1-0.39 – 840 bp R: CGATCCAAATGATCCTGAAGA 7BL2-0.38–0.63 7DL1-0.14–0.30 10 TNAC1903 F: TCGCTTCTTCTGCTTGTTCTT C-7AL1–0.39 TaqI 920 bp* R: CTGCTACTAGGCCACCCAAA C-7BL2–0.38 7DL1-0.14–0.30 11 TNAC1926 F: CGTCAGCTACAGCGACATCTA C-7AS8–0.45 TaqI 700 bp* R: AACTTGAGCAGCGTGGTGTT 7BS2-0.27–1.00 7DS3-0.15–0.36 12 TNAC1943 F: GCTGCTATGGTCCACGAATTA 7AS5-0.59–0.73 HaeIII 600 bp* R: AGAGTATCGTATCCGGGCAAT 7BS2-0.27–1.00 7DS4-0.73–1.00 13 TNAC1957 F: TCAACATTTGCAGGATTGTCA 7AL21-0.74–0.86 – 730 bp R: TTTCACAGGAACCTCTGCATC 7BL10-0.78–0.84 7DL2-0.61–0.82 The primers and the location in wheat bin map were referred to Ishikawa et al. [18]. The star indicated the Vb7 specific bands were polymorphic to 7V band patterns reflected the large genomic divergence be- D. villosum and tetraploid D. breviaristatum evolved in tween the D. villosum and D. breviaristatum chromo- parallel from an ancestral species. Recently, we used the somes. Liu et al. [14] used FISH probed by a ribosomal pDbC2 probe to hybridize to tetraploid D. breviaristatum DNA sequence and proposed a hypothesis that the diploid and diploid D. villosum chromosomes, and found more Li et al. Molecular Cytogenetics (2016) 9:6 Page 6 of 9

Fig. 3 PCR using PLUG primers TNAC1782/HaeIII (a), TNAC1811/TaqI (b), TNAC1812/TaqI(c) and TNAC1867 (d). The stars and arrows indicate the D. breviaristatum specific bands in TDH-2 and D2139, respectively

Ty3-gypsy retrotransposon copy numbers in centromeric pHv62-1 can hybridize D. villosum chromosomes in regions of D. villosum than those in D. breviaristatum TDV-1 (Fig. 1e). It confirmed that the high tandem repeat [24]. In the present study, we compared D. breviaristatum sequences present largely in telomeric heterochromatin and diploid D. villosum chromosomes through the distri- regions of D. villosum as reported by Li et al. [17]. How- bution of FISH signals of Oligo-pSc119.2, Oligo-pTa535, ever, FISH revealed that Oligo-pHv62-1 was absent in D. Oligo-(GAA)7 by sequential multicolor-FISH (Fig. 1) and breviaristatum chromosomes of TDH-2 (Fig. 1f). This the molecular markers (Table 1) by PCR. The results sug- significant amplification of different types of repetitive se- gested that strong evolutionary divergence involving copy quences between the D. villosum and D. breviaristatum number of repeated sequences and nucleotide sequence chromosomes may be related to adaptation of the plant rearrangement may have occurred among Dasypyrum species to their environments [3]. The cytogenetic and species during species evolution. Moreover, the Oligo- molecular markers which are species-specific can be used

Fig. 4 The spikes morphology (a) and leaf response to stripe rust (b) of the lines Li et al. Molecular Cytogenetics (2016) 9:6 Page 7 of 9

to identify and characterize the introgression of D. villosum from D. breviaristatum to wheat, we identified the two D. and D. breviaristatum chromosome segments into a wheat breviaristatum chromosomes addition lines Y93-1-A6-4 background. and Y93-1-6-6, which showed novel resistance to powdery Rapid genomic and epigenomic changes have been mildew isolates and (pathotype TTKSK) commonly found in some newly synthesized wheat- [11]. Molecular marker and GISH analysis revealed that alien amphiploids [25, 26]. Alterations of alien chromo- those introduced D. breviaristatum chromosomes were somal structure in wheat background have also been rearranged chromosomes involved groups 2, 6 and 7. Re- described especially wheat-rye chromosome addition, cently, Li et al. [12] reported a wheat - D. breviaristatum substitution and translocation lines [27, 28]. However, substitution line D11-5 possessing a pair of 2Vb chromo- the variations in the karyotype of wheat chromosomes somes which had replaced wheat 2D. Based on the FISH have been less reported. Recently, Fu et al. [29] re- analysis, we found that the 2Vb chromosome in line D11- ported that pSc119.2 FISH signals could be observed at 5 was identical to chromosome Vb2 of TDH-2 (Fig. 1). the telomeric regions of 3DS arms which was not ob- We thus suggest that chromosome Vb2 can be provision- served in the current material, and structural variation ally assigned to linkage group 2, subject to confirmation and abnormal mitotic behavior of the 3D chromosome using other genetic markers. were detected in the selfed progeny of wheat “MY11”- In the present study, we identified line D2139 which rye 6R monosomic addition line. Furthermore, Fu et al. contained a pair of D. breviaristatum chromosomes con- [30] reported the occurrence of 14 chromosomal rear- firmed herein to be “7Vb”. This disomic substitution line rangements in wheat -rye hybrids. Our studies found D2139 may be potentially useful germplasm for agro- that chromosomes 1B, 2B and 7A of the wheat- D. breviar- nomic traits including enhance spike length and the istatum partial amphiploid TDH-2 (Fig. 1), and chromo- stripe rust resistance from the D. breviaristatum 7Vb into somes 1D and 3D of the 7Vb addition lines D2139 (Fig. 2) the wheat genome using marker-assisted chromosome en- showed apparent structural changes revealed by FISH gineering [37]. The divergence between the individual D. patterns compared to the parental lines. Patokar et al. [31] villosum 7V and D. breviaristatum 7Vb chromosomes was characterized several novel wheat-Thinopyrum bessara- revealed by FISH and molecular markers in wheat back- bicum recombinant lines carrying intercalary translocations ground, which will provide the basis for future detailed and did not report any observable wheat chromosomal re- comparative genomics analysis. Guo et al. [38] compared i arrangements using FISH. It is likely that the distant species chromosomes 7el1,7el2,7E(e),and7E derived from differ- of genera and Dasypyrum may induce such struc- ent Thinopyrum species by molecular and cytological tural changes while present in a wheat background, while methods. In a similar manner, we intend to create hybrid chromosomes derived from Thinopyrum species may not populations between wheat- Dasypyrum 7 V and 7Vb have the same effect due to their close relationship to addition lines for further and direct localization of genes wheat [32]. Thus, we suggest that the introgression of on these alien chromosomes. chromosomes from closely related species may not lead to the significant structural changes of wheat chromo- Conclusions somes, although the introduction of closely related Aegilops In summary, the different FISH patterns between D. chromosomes causes massive deletions of wheat chromo- breviaristatum and D. villosum chromosomes were ob- somes [33], which were mainly useful for physical mapping served clearly by using different repetitive sequences as of genes. There is the other possibility that the recipient probes, which allows to identify the individual Dasypyrum wheat genotype may also increase the chromosomal re- chromosomes in wheat background. The changes of FISH arrangement with visible changes of representative repeats. patterns of wheat chromosomes were induced by the in- Taking advantage of fast multicolor FISH methods [15], we duction of D. breviaristatum to wheat. The D. breviarista- recently identified some chromosomal changes in high tum specific molecular markers can be used to assign the yielding elite cultivars originating from wheat distant homologous group of D. breviaristatum to wheat. We iden- hybridization. The association between the visibly rear- tified wheat- D. breviaristatum chromosome 7Vb addition ranged wheat chromosomes and the yield or disease re- line with novel stripe rust resistances will be potential sistances are being verified for breeding purpose. useful for wheat breeding. The molecular and cytogenetic D. villosum chromosomes are known to contain genetic markers will assist to trace the D. breviaristatum chromatin variability of value for incorporation into wheat. At least in wheat background. three sets of D. villosum chromosomes addition lines in wheat background have been developed [34–36]. Novel Methods genes including disease resistance and quality-related Plant materials characters have been found in different wheat- D. villosum D. breviaristatum accession PI 546317 was obtained derived lines [7]. With the aim to transfer novel genes from the National Small Grains Collection at Aberdeen, Li et al. Molecular Cytogenetics (2016) 9:6 Page 8 of 9

Idaho, USA. The wheat– D. breviaristatum partial am- Competing interests phiploid TDH-2 (genome AABBVbVb) was as described The authors declare that they have no competing interests. by Yang et al. [10]. The accession of Dasypyrum villosum Authors’ contributions TA10220 and the Chinese Spring- D. villosum chromo- GL and ZY designed the study. GL, DG, HZ, JL conducted the experiments. some 7 V addition line TA7683 [39] were obtained from HW, SL and JM helped to conduct experiment and data analysis. GL and ZY Dr. Bernd Friebe of Wheat Genetic and Genomic Re- participated in paper writing. All authors read and approved the final manuscript. sources Center at Kansas State University, Manhattan, Acknowledgement KS, USA. The T. turgidum cv. Jorc-69- D. villosum am- We particularly thank Dr. I. Dundas at University of Adelaide, Australia, for phiploid TDV-1 (genome AABBVV) was developed and reviewing the manuscript. We thank the National Natural Science Foundation of China (No. 31171542), and Sichuan wheat breeding community for the provided by Prof. Hua-Ren Jiang at Sichuan Agricultural financial support. University, China [40]. Line D2139 was obtained from a Received: 3 December 2015 Accepted: 19 January 2016 BC1F5 generation of the crosses between wheat cultivar ‘Mianyang 11’ (MY11) and TDH-2. References Fluorescence in situ hybridization (FISH) 1. Frederiksen S. Taxonomic studies in Dasypyrum (). Nord J Bot. Seedling root tips were collected and then treated with 1991;11:135–42. 2. Gradzielewska A. The genus Dasypyrum-part 1. The and relationships nitrous oxide followed by enzyme digestion, using the within Dasypyrum and with species. Euphytica. 2006;152:429–40. procedure of Han et al. [41]. The synthesized oligo- 3. De Pace C, Vaccino P, Cionini PG, Pasquini M, Bizzarri M, Qualset CO. Dasypyrum. nucleotide probes Oligo-pSc119.2, Oligo-pTa535, Oligo- In: Kole C, editor. 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High frequency of centromere inactivation • Inclusion in PubMed and all major indexing services resulting in stable dicentric chromosomes of maize. Proc Natl Acad Sci U S A. 2006;103:3238–43. • Maximum visibility for your research

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